System and method for performing in vivo imaging and oxymetry and FT microscopy by pulsed radiofrequency electron paramagnetic resonance

ABSTRACT

A system for performing pulsed RF FT EPR spectroscopy and imaging includes an ultra-fast excitation subsystem and an ultra-fast data acquisition subsystem. Additionally, method for measuring and imaging in vivo oxygen and free radicals or for performing RF FT EPR spectroscopy utilizes short RF excitations pulses and ultra-fast sampling, digitizing, and summing steps.

BACKGROUND OF THE INVENTION

This invention describes a fast response pulsed Radiofrequency (RF)Electron Paramagnetic Resonance (EPR) spectroscopic technique forin-vivo detection and imaging of exogenous and endogenous free radicals,oxygen measurement and imaging and other biological and biomedicalapplications.

The main emphasis is the use of low dead-time resonators coupled withfast recovery gated preamplifiers and ultra fastsampler/summer-summer/processor accessory. Such a spectrometer will bepractical in high resolution detection and imaging of free radicalspossessing narrow line widths. This method avoids factors compromisingthe imaging speed and resolution inherent in the existing ContinuousWave (CW) EPR imaging methods, where modulation and saturationbroadening and artifacts of object motion are problems.

It is also possible to perform Fourier imaging and hence to produceimage contrasts based on relaxation when using special narrow-line freeradical probes.

The response of tumors to radiation therapy and chemotherapeutic agentsdepends upon the oxygen tension. Hence, for an effective cancer therapy,measurement of molecular oxygen in tumors is vital. Also in generalmedicine measurement of the oxygen status of ischemic tissue incirculatory insufficiency, be it acute as in stroke or myocardialinfarction, or chronic as in peripheral vascular disease associated withnumerous diseases such as diabetes, hyperlipidemias, etc., becomes animportant tool for assessment and treatment of diseases. Although avariety of techniques are available for measuring oxygen tension inbiological systems, polarographic technique is perhaps the one mostwidely used in clinical applications. However, this is an invasivetechnique. Besides patients' discomfort, the tissue damage caused by theprobe electrodes leads to uncertainty in the values measured, especiallyat low oxygen concentration (<10 mm Hg).

Magnetic Resonance Imaging (MRI) enjoys great success as a noninvasivetechnique. NMR imaging based on perfluorinated organic compounds hasbeen used to study blood oxygenation of animal brains. Binding of oxygento hemoglobin is also used as a marker in MRI of human brains to monitoroxygenation changes. However, these techniques lack sufficientsensitivity for routine applications.

Overhauser magnetic resonance imaging (OMRI), based on the enhancementof the NMR signal due to the coupling of the electron spin of anexogenously administered free radical with the water protons, is alsoattempted for in-vivo oxymetry. Here again the sensitivity is limited,since the organic free radicals used have low relaxivity since theydon't possess the free sites for water binding as in the case ofgadolinium based contrast agents. The Gd based contrast agents, however,have relaxation times that are too far for efficient spin polarizationtransfer. On the other hand, EPR oxymetry compared to MRI or OMRI isvery sensitive for oxygen measurements, since it is based on the directdipolar interaction of the paramagnetic oxygen molecule with the freeradical probe.

EPR is generally performed at microwave frequencies (9 GHz). The use ofmicrowave frequency results in substantial tissue heating, andunfortunately, severely limits tissue penetration. Low-frequency EPR hasbeen attempted to achieve better tissue penetration. All of thesestudies except the last one (from this lab) were done using theContinuous Wave (CW) method.

Although low-frequency EPR offers the potential for greater in-vivotissue penetration, its use in continuous wave-based methods is severelylimited by lack of sensitivity resulting from the physically imposedBoltzmann factor. Furthermore, sensitivity enhancement by signalaveraging as done with CW methods, may not be effective since CW methodsare band limited. Pulse EPR techniques, however, as presented in thisapplication, advantageously utilize the very short electron relaxationtimes to rapidly enhance the signal to noise ratio, which immediatelyleads to speed and sensitivity advantage in pulse EPR detection andimaging.

Further, the absence of any modulation in the method leads to true linewidths, whereas in the CW methods finite modulation can, in the case ofnarrow lines, lead to artifacts and, therefore, can severely limit theresolution achievable. Power saturation is another factor that greatlylimits the resolution when detecting and imaging narrow line systems.Also for in vivo studies, any movement of the subject being studiedposes severe problems in CW methods. Further, relaxation weightedimaging for contrast mapping is feasible mainly with the pulsed methods.Most of these advantages of pulse techniques over CW method are wellestablished in MRI.

Application of pulse techniques to EPR has serious limitations. The veryadvantage of short relaxation time, which can in principle lead tovirtual "real time" imaging, poses a challenge to the state of the artelectronics for ultra fast excitation and data acquisition. Instrumentaldead-time problems become very severe, especially at low frequencies,since the ringing time constant, t=2 Q/w (where Q is the resonatorquality factor and w is the carrier frequency), allows acquisition ofsignals only after a significant interval following excitation, whichcan lead to loss of sensitivity.

The current invention addresses all of these problems and outlinespulsed EPR methodologies at radiofrequency region for in-vivo imaging offree radicals and oxygen measurement and imaging of oxygen usingsuitable paramagnetic agents.

Apart from oxygen measurements, using appropriate free radical probesone can perform rapid imaging to map out blood vessels (for example,cardiac and cerebral angiography), study tissue characteristics and freeradical metabolic intermediates in situ with or without using spintraps. Use of free radical probes also provides the ability to useadministered paramagnetic contrast agents for imaging both normal anddiseased tissues.

This invention has additional advantages as follows. Firstly, themagnetic field used is only about of 10 mT, orders of magnitude lessthan in MRI. Secondly, the sensitivity achievable is much higher thanOMRI. Lastly, sensitivity enhancement, image resolution and imagingspeed and T1 and T2 weighted imaging modalities are far superior to CWRF EPR.

SUMMARY OF THE INVENTION

According to one aspect of the invention, a pulsed EPR FT imaging andspectroscopy system includes an excitation system for forming 20 to 70nanoseconds RF excitation pulses of about 200 to 400 MHz. A gated dataacquisition system with very low dead time generates EPR responsesignals. A pulse sequence with a repetition rate of about 4 to 5microseconds is sampled and summed to provide a signal having a highsignal-to-noise ratio.

Another aspect of the present invention is a novel RF FT EPR techniquefor measuring oxygen tension in vivo in biological systems inconjunction with a suitable narrow-line oxygen-sensitive free radical (See Anderson, G. Ehnholm, K. Golman, M. Jurjgenson, I. Leunbach, S.Peterson, F. Rise, O. Salo and S. Vahasalo: Overhause MR imaging withagents with different line widths, Radiology 177, 246 (1990);Triarylmethyl radicals and the use of inert carbon free radicals in MRI,World Intellectual Property Organization, International Bureau,International Patent Classification A61K 49/00, C07D519/00, C07B61/02//C07D 493/04, International Publication, No. WO 91/12024,(22.08.1991) and for the detection and imaging of endogenous andexogenous free radicals. The subject of study, placed in a suitableresonator of low Q, high filling factor and coupled to a RF pulseexcitation system (vide infra) is given an injection of the free radicalprobe and immediately thereafter it is subjected to an intense short RFpulse. The time response of the RF signal, which will be oxygendependent and/or the signature of the free radical present, is acquiredusing a very fast acquisition system. The signal-to-noise ratio isenhanced to an extent of 60 dB in just one second by coherent averagingusing an ultra fast averager (vide infra). The spatial resolution inthree-dimension is obtained by using a set of three-axis gradient coilsystem.

According to another aspect of this invention, stochastic excitation orpseudo stochastic excitation with subsequent Hadamard transformationwill be used where a large bandwidth is to be excited, instead of usinga compressed high power pulse. This will avoid sample heatingconsiderably, because the power required for stochastic excitation is atleast an order of magnitude less than in the conventional pulsedtechniques. The principle and application of Hadamard transformation iswell documented and illustrated in NMR spectroscopy and imagingliterature. The RF carrier is modulated by a pseudorandom binarysequence which is generated in a shift register and the values of thesequence are used to modulate the RF phase for each sampling interval tby +90° or -90°. The pseudo-noise sequence thus generated will berepeated in a cyclic fashion after a given number of values. Theacquisition of the response and phase cycling follow standardprocedures. A Hadamard transform of the response produces the FID freeinduction decay which, upon complex Fourier transform, yields a spectrumor a single projection when gradients are present.

According to another aspect of the invention, by using free radicalprobes of long relaxation time gradient switching can be used to performslice-selective EPR tomography as in MRI, as well as all other imagingmodalities used in MRI. Additionally, high gradients can be used toperform EPR microscopy.

Other advantages and features of this invention will be made apparentfrom the following drawings and descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an overall block diagram of the spectrometer and imager;

FIG. 2A is a schematic diagram of the phase-shifter used in a preferredembodiment;

FIG. 2B is a schematic diagram of the high-speed gates used in apreferred embodiment;

FIG. 2C is a schematic diagram of the diplexer used in a preferredembodiment;

FIG. 2D is a schematic diagram of the gated preamp used in a preferredembodiment;

FIGS. 2E-F are schematic diagrams of a Q-circuit and an equivalentQ-circuit, respectively, utilized in the resonator of the preferredembodiment;

FIG. 2G is a schematic diagram of the quadrature detector used in apreferred embodiment;

FIG. 2H is a layout diagram of the ultra-fast data acquisition subsystemused in the preferred embodiment;

FIG. 2I is a block diagram of the summing part of the ultra-fast dataacquisition subsystem of FIG. 2H;

FIG. 3 is a timing diagram relating to the operation of the preferredembodiment;

FIGS. 4A-D are timing diagrams for using the system to implement aHadamard excitation scheme;

FIG. 5 is a flow chart giving the details of generating an image.

DESCRIPTION OF THE PREFERRED EMBODIMENT

FIG. 1 is a block diagram of the spectrometer/imager. RF power from aHewlett-Packard (Palo Alto, Calif.) signal generator model HP8644A, 1 issplit by a two-way-zero degree power splitter (model ZSC-2-1W,Minicircuits, Brooklyn, N.Y.) 2 into two ports, one serving thereference arm and the other the transmitter side. The reference side isgated using RF gate 4b. The required gate timing is provided by pulsegenerator 6 in the form of a cluster of four Digital Delay Generators(model 535, Stanford Research Systems, Sunnyvale, Calif.). Forsynchronization, the first of the delay generators utilizes the systemclock generated by the RF signal generator 1, a trigger input (10 MHz),thereby reducing the jitter of the delay outputs to less than 25 ps rms.The time base drift between the various delay generators is eliminatedby daisy chaining the reference output of the first DG535 with thereference input on the other DG535's. An appropriate level of referencesignal for mixing is selected using the variable attenuator 5a.

The other arm of the splitter is directed through a 0°/180° phaseshifter 3 which can be software controlled using timing pulses from thegenerator 6. The transmitter pulse is gated through 4a, a gating circuitfurther amplified by a home-made RF amplifier 7a (25 db) and stillfurther amplified by a power amplifier (ENI 5100L, 100 W) 7b. Theoptimization of the RF power level is accomplished using a set ofattenuators 5b and 5c. The amplified pulses are coupled with thediplexer T/R switch 9 through a pair of crossed diodes 8 for protectionfrom the reflected pulse generator. The diplexer switch 9 receives thetiming signal from 6 and the RF pulse is delivered to the resonator 12.

The magnetic induction response from the object in the resonator isfirst taken through a specially designed gated preamplifier 10 with alow-noise high-gain (45 dB) capability and a very short saturationrecovery time. The preamplifier gate switching is also controlled bypulse generator 6. The output of the preamplifier is further amplifiedusing amplifiers 11 and 7c with suitable attenuation in between byattenuators 5d and 5e to avoid saturation.

The reference signal from 4b and the amplified induction signal from 7care mixed using a double-balanced quad mixer 17. The real and imaginaryparts are passed through two identical low-pass filters 18a and 18b,before sampling, using a specially designed ultra-fastsampler/summer/averager 19. The averaged signal is processed in aSilicon Graphics computer 20 which also controls the overallspectrometer/imager as shown by the bus connection in FIG. 1.

The resonance condition is set by changing the current in the DC magnet13 using the power supply 14, which is addressed by the computer.

For imaging, the spatial/spectral distribution of the spin is frequencyencoded by using a set of 3- axes orthogonal field gradient coils 15.The gradient steering is done by software control of the gradient powersupply 16. The overall process of generating the image/spectrum issummarized in FIG. 6.

The various components/modules depicted in FIG. 1 will now be described.In the preferred embodiment, the RF signal generator 1 is a HewlettPackard model 8644A--Synthesized Signal Generator and the splitter 2 isa Minicircuits ZSC-2-1W (1-650 MHz).

The phase shifter 3 is depicted in FIG. 2A and has been designed andbuilt for the removal of systematic noise. A gating pulse C provided bythe pulse generator may have either negative polarity (to induce a 180°phase shift) or positive polarity (to induce a 0° phase shift) where thepolarity is controlled by the host computer 20.

RF from the transmitter can, despite the good isolation between thetransmitter (Tx) and receiver (Tx) provided by the diplexer and thevarious gatings, leak into the receiver. This leakage can arise frompulse breakthrough while the transmitter is on and/or from directradiation into the receiver from within the spectrometer's electronics.This results in unwanted dc output from quad mixer 17. If uncorrectedthis can lead to a large dc bias and result in a spurious spike at zerofrequency upon Fourier Transformation.

With the phase shifter set at 0° phase a group of FIDs, say 1000, isaccumulated. Then the phase of the RF pulse is changed 180° by a pulsegiven from the pulse generator 6, and another 1000 FIDs are accumulated.The resultant FID signals are unaffected, except for the change in sign,and thus these are subtracted from the previous group leading to a totalcollection of 2000 FIDs. The unwanted dc biases, from the RF leakage andthe amplifiers' drift, and other systematic noises do not change in signand thus they get subtracted. Thus, the phase shifter, besides removingthe unwanted systematic noises, also helps to reduce the data collectiontime by half.

The gates 4a are depicted in FIG. 2B and should possess a very highon-off ratio (typically 100-120 dB) to avoid any RF leakage through thegate to the sample. Further, the rise time of the gate should be veryshort, since pulses of the order of 10 to 20 ns are used in RF FT-EPR incontrast to pulses of tens of microsecond or millisecond in NMR. Even atwo ns rise time will make a 10 ns pulse and can distort the desiredsquare wave pulse. Also, the gate opening and closing glitches should beminimal to avoid any amplification by the power amp 7b. To meet thesedemands of ultra-fast excitation needed for the RF FT-EPR, the specialgates depicted in FIG. 2B have been designed and built.

The attenuators 5 are Kay Electronics model 839, and the pulse generator6 is a cluster of Stanford Research Systems DG535 four-channel digitaldelay/pulse generators; the RF Amplifier 7a is a 10-400 MHz, 5 dBm inOP-AMP +25 dBm out--Motorola MHW 590; the RF Power Amplifier 7b is anENI Model 5100L Watt, 50 dB; and the cross-diodes 8 are IN 9153 diodes.These cross diodes 8 disconnect the transmitter from the probe (tankcircuit) and the preamplifier during the receiving mode to reduce thenoise.

The diplexer 9 is depicted in FIG. 2C. A major requirement for asensitive RF FT-EPR spectrometer is to design a suitable technique tocouple the transmitter, probe and receiver. During the transmit cyclehigh RF power of the transmitter should be delivered to the sample inthe probe without damaging or overloading the sensitive receiver, andduring the receiving mode any noise originating from the transmittermust be completely isolated. This is not trivial since the EPR signal ofinterest is in the microvolt range whereas the transmitter signal ishundreds of volts.

Further, in contrast to NMR, the very short relaxation time of EPRdemands very fast closing and opening of these gates. In FIG. 2C, thediplexer gating pulse is generated by the pulse generator 6. The RFexcitation pulse is received at Tx and is coupled to the probe by thediode switch when the diplexer gating pulse is asserted. Tx is isolatedfrom the probe and Rx when the diplexer pulse is not asserted. ALAMBDA/4 cable delays the arrival of the EPR response pulse until thegate preamplifier 10 receives the gating pulse.

Within 15 ns of the closing of the transmitter the transmitter, signalcomes down to the level of the background noise.

The gated preamplifier 10 is depicted in FIG. 2D and has a gain of 46dB. An important problem faced in RF FT-EPR is the overload recovery ofthe receiver system, especially at the front end, namely, thepreamplifier. Even small glitches after the close of the Tx cycle or thering-down signals can easily overload and "paralyze" the preamplifier.Hence, the preamp should have a very fast rise time and high gain.Otherwise the weak FIDs can get clobbered by the overload recoveryproblems. In NMR the recovery can be in micro seconds, but the shortrelaxation times of EPR demand nano second recovery. Otherwise it is notpossible to recover the signals as soon as the subject of study isexposed to the RF pulse.

Specially designed cascaded amplifiers for high gain and fast recoveryare needed, especially if the preamp can be gated; then theabove-mentioned glitches following the Tx can be avoided. Since thereare no such gated preamplifiers available, a gated preamplifier 10 hasbeen designed and built with a fairly wide dynamic range (gain 46 dB),low noise and a very fast recovery time of 2 to 5 nano seconds. The gateof the preamp is opened 5-10 ns after the transmitter pulse to avoidoverload saturation. The gate pulse is provided by the pulse generator.

As depicted in FIG. 2D, four monolithic amplifiers, e.g., the MAR seriesfrom Minicircuits, in Brooklyn, N.Y., are cascaded. Diode switchesbetween the amplifiers are switched by a gating pulse generated by pulsegenerator 6. Other switches instead of diode switches could be used.Amplifier 11 is a MITEQ Amplifier Model 2A0150

The time constant associated with a resonant circuit is given by

    TAU=Q/PI*NU

Where NU is the resonance frequency following a Tx pulse of about 100 Vinto the resonant circuit, at least about 20 time constants are requiredfor ringing down the decay to the level of a small but measurable FIDsignal of about 2 micro volt. As seen from the above equation theringdown time constant is inversely proportional to the frequency. Inthe case of EPR at conventional frequencies (9 GHz), this time constantis much lower than it is at the RF frequency.

Although TAU can be reduced by lowering the quality factor Q, thesignal-to-noise ratio of the EPR signal is proportional to Q. Hence, Qcannot be compromised too much, especially so at low frequency where thesignal to noise ratio is already limited by the Boltzmann factor. Also,this problem in RF FT-EPR is much more severe than the NMR due to thevery short decay of the FID from the EPR signal. Hence, the resonatorshould have a short recovery time to collect the FID. Since the FIDdecays exponentially, even a small gain in the ringing time minimizationcan make a large difference in acquiring the signal. We have adopteddifferent approaches to solve this problem depending upon the sample ofstudy.

Probes with equal subcoils in parallel

Since high Q coils cannot be used at low frequencies we adopted otherstrategies to improve the sensitivity. The S/N ratio depends on otherfactors such as the filling factor (F) and volume (V) of the coil. Thisdependence is given by

    S/N=√(F/QV)

The coil volume was increased by adding solenoidal coil segments andwiring them in parallel. This coil with segments in parallel has lessinductance than a single coil of the same size and thus it is possibleto make a large size coil to accommodate more sample for a particularfrequency, thereby increasing the S/N ratio.

We have reduced the Q to optimum values, depending upon the relaxationtimes of the free radical probes used, by using the overcoupling methodrather than Q spoiling, since the signal intensity is greater in theovercoupling method by a factor of 2 as given by

    S overcoupled/S spoiled=√(2 β/1+β)

where β is the coupling constant.

When sensitivity requirements demand high Q, dynamic Q-switching 36 canbe used to cut down the resonator ringing time. Schematics of aQ-switching circuit are given in FIGS. 2E-F. The capacitor c₂ is usedfor tuning and C_(m) for matching. A nonmagnetic GaAs beam lead PINdiode from M/A-COM (Burlington, Mass.) is used for Q-switching. In thenormal mode of operation R_(p) is effectively the small forward biasresistance of the PIN diode. Q-switching is done by sending a shortpulse (20 ns) immediately after the transmit RF pulse. DuringQ-switching R_(p) is the large reverse bias resistance of the PIN diodein parallel with R_(R). By selecting optimum C₁, C₂, C₃ and Rp the totalresistance of the network is maximized to minimize the ringdown timeconstant,

    TAU min=2L/(R max+RL)

where Rmax is given by

    R max=((Rp) opt/2 (C1/C2+1)2

Thus, during the switching pulse, the Q of the system gets low, therebyenabling faster ring down. However, after the switch pulse the Q becomesnormal in the receive cycle for greater sensitivity.

Active damping for bandwidth enlargement

To study relatively large size objects the bandwidth of excitationincreases. In NMR, even a bandwidth of 70 KHz is relatively very large.However, in EPR a bandwidth of 50-70 MHz may be needed. In principle,bandwidth enlargement can be achieved by placing a resistor in parallelwith the tuned circuit. This passive damping, however, will degrade thesignal. Hence, active damping can be used to enhance the bandwidth andto bring down the ringing time. According to this procedure, a preampwith negative feedback is employed to enhance the bandwidth withoutseriously degrading the signal.

Other types of resonators such as loop-gap or bird-cage types are used.These are designed to have low Q and are matched by overcoupling oractive damping to enhance the bandwidth and to bring down the ringingtime.

One or two turn surface coils are also used for topical applicationswhere the size of the subject is too large to be accommodated inside theresonator

Another type of resonator used is of a miniature catheter type forangiographic applications.

The DC magnet 13 is a Magnet GMW Model 5451; the magnet power supply 14is a Danfysik System 8000, Power Supply 858; the gradient coils 15 are(a) specially designed air-cooled three axes gradient coils for 3Dimaging and (b) surface gradient coils 38 for organ specific imaging.The gradient coils power supply 16 is an HP 6629A+ specially designedmicrocomputer controlled relay system. The quad mixer 17 is depicted inFIG. 2G and the low-pass filters 18a and b are specially designed.

The sampler/summer/averager 19 will now be described with reference toFIGS. 2H and I.

The magnetic induction response of the system of study to the excitingRF pulse is generally weak. To improve the signal-to-noise ratio it isnecessary to carry out the signal averaging of the transient response.This is done by first digitizing and then summing the digitized data.The large line width (MHz in contrast to Hz or KHz in high resolution orsolid state NMR) and the short relaxation times (nanoseconds in contrastto micro- or milliseconds in NMR) encountered in EPR cause severeproblems in the design and construction of suitably fast dataacquisition systems for EPR imaging.

High speed digitizers with sampling frequencies up to even GHz range arenow commercially available. However, these devices are generallysuitable for capturing single-shot events and the summing speed of thedigitized data in these instruments for data accumulations is very slow.Such slowness prohibits one from taking advantage of the very shortelectron spin relaxation time and thereby limits the ability to improvethe S/N ratio by carrying out a large number of coherent averages in ashort period of time. Hence, we have utilized an ultra-fastsampler/summer/averager to enhance the speed of data collection forimaging. As shown in the block diagram of this system in FIG. 2H, itconsists of three modules: a sampler, a summer and a processor.

The sampler contains four high-precision TKA10C 500-MSPS analog todigital converters. It has a vertical resolution of 8 bits, with asensitivity of +/-250 mV full scale. The sampler also has an overloadprotection of +/-6 volts. The sampler has two channels with a maximuminterleaved sampling rate of 1 GS/s per channel or 2 GS/s if it is usedin a single channel mode.

The amplifier Plug₋₋ Ins provide gain, offset and calibration signalinjection for the input signal and provide sufficient drive capabilityfor the ADCs. The signals I and Q from the quad detector are shown asSIG1 and SIG2.

Calibration and correction circuitry are provided to correct AC and DCerrors at their source. A trigger controller provides triggeringcapability from the external source. In the Level Triggering mode, thetriggering circuitry is enabled when the ARM input is at a TTL highlevel (given by the pulse generator) and the ACQUIRE signal has beenreceived from the processor. The sampler then digitizes the data (FID)and sends it in eight parallel data streams (each at 250 MS/s) on thegigaport.

The gigaportout from the VX2004S sampling module provides data, clock,and control and monitoring signals to the VX2001 signal averager. Thereare four channels, each providing a 16 bit stream of data. Channels Aand B provide the digitized data of signal 1 (Q-of the quadratureoutput) and C and D that of signal 2 (I of the quadrature output). Theinput FIFO (First-In-First-Out) memories buffer the data from thegigaports. The FIFO memories can accommodate a record length of 8192samples for each of two sampler channel pairs (A/B and C/D). A detailedblock diagram of one of channel is given in FIG. 2I.

The summing process begins when the processor activates a control signal`P₋₋ ACCUIRE`. In response to this the summer/averager activates thesampler which in turn starts to send the digitized data over the fourchannels. The VX2001V sums the digitized waveform data words and thenreactivates the sampler to initiate the next digitizing cycle. Thisprocess repeats until a programmed number of FIDs are summed. Thisprogrammable number is a 24-bit word and hence more than 4 millionaverages can be done without transferring the data to the processor.

The summation process operates in conjunction with the digitizationprocess by the sampler when the sampler operates in Pre-Trigger mode.The summing process begins when the first words have been loaded intoall of the input FIFO memories. Thus, the summing process effectivelyoverlaps the digitization process since it does not have to wait untilthe input FIFO's loading process has been completed. FIDs with a recordlength of 1024 for both the signals at 1 GSPS can be summed at a rate of230 KHz. (retrigger period of approximately 4.3 ms.) The data outputfrom the summer is 32 bits wide and passed in sequence to the VX2000Pprocessor as two 16-bit words.

The VX2000P processor module contains:

A Motorola 68340 microprocessor with the support of an integral2-channel DMA controller, 4 MB DRAM, 128 KB EPROM, 2 MB Flash EEPROM, 2integral timers and 2 serial I/O channels;

an IEEE488.2 GPIB port for interface with host computer;

a graphic processor with 2 MB of DRAM, 512 KB of VRAM, a VGA compatiblevideoport providing 1024*768*4 graphics;

two channels of data acquisition memory capable of acquiring data fromthe summer at a rate of 500 MB per second via the front panel gigaportconnector; and

a gigaport connector that supplies interfaces between the processor andthe other modules;

A high speed parallel output port for the delivery of data to theexternal device (Host Computer/Image processor).

The large on board memory and the video graphics allow collection andprocessing of more than 40 data projections before downloading the datato the host computer.

Thus, the large bandwidth of the sampler, the summing speed, the largedynamic range of the summer/averager, on-board data memory of 16 MB RAMand fast data transfer of the processor module provide an ultra-fastDAS, enabling increased sensitivity and imaging in a short time.

The computer 20 is a Silicon Graphics IRIS-4D.

FIG. 3 is a timing diagram depicting the pulses generated by pulsegenerator 6 to control the various elements in the system of FIG. 1 fora one pulse experiment. A transmit gating pulse 30 is generated tocontrol the high-speed gate to transmit an RF pulse having a duration ofabout 10 to 70 nanoseconds. For larger samples the length of the pulsecould be extended up to 100 nanoseconds.

The timing of the diplexer gating pulse 32 is best understood byconsidering the shape 34 of the RF pulse generated by the poweramplifier 7a. The diplexer gating pulse 32 is asserted at the trailingedge of the transmit gating pulse 30. There is about a 25 nanoseconddelay caused by the power amplifier 7a before the RF pulse reaches thediplexer. The diplexer gating pulse also extends about 30 nanosecondsbeyond the end of the RF pulse. The receiver, preamp, andsampler/averager gating pulses 36, 38, and 40 are all asserted at thetrailing edge of the diplexer gating pulse.

As described above, this timing is critical to keep the gatedpreamplifier 10 from saturating. The magnitude of the RF pulse is muchgreater than the magnitude of the EPR response signal. Thus, anytransients or glitches resulting from ringdown in the resonator wouldoverwhelm the preamp 10 and cause saturation. Recovery from saturationin a cascaded amplifier is very slow and the system would becomeinoperative.

Thus, the 30 nanosecond delay between the end of the RF pulse and theleading edge of the preamplifier gating signal allows for damping oftransients and glitches and prevents preamplifier 10 from saturating.The preamplifier 10 generates an EPR response signal which includes theRF carrier signal and information relating to EPR parameters andresonant frequencies.

The quadrature mixer 17 processes the EPR response signal to generate anEPR parameter signal which is further processed to determine EPRparameters such as relaxation time and resonant frequencies.

As described above, in practice many transmit pulses are generated andthe corresponding EPR responses summed to improve the signal-to-noiseratio. Typically, the transmit gating pulses 30 are generated at arepetition rate of 4 to 5 microseconds. This allows summing betweenpulses which takes about 4 microseconds. For large gradient fields therepetition rate could be slowed.

The pulse sequence for stochastic excitation or pseudostochasticexcitation is depicted in FIGS. 4C and 4B. This excitation sequence withsubsequent Hadamard transformation will be used instead of a compressedhigh power pulse where a large bandwidth is to be excited. This willconsiderably avoid sample heating, because the power required forstochastic excitation is at least an order of magnitude less than in theconventional pulsed techniques. The principle and application ofHadamard transformation is well documented and illustrated in NMRspectroscopy and imaging literature.

The rf carrier is modulated by a pseudorandom binary sequence, asdepicted in FIG. 4A, which is generated in a shift register or acomputer program, and the values of the sequence are used to modulatethe rf phase for each sampling interval DELTA(T) by +90° or -90°, asdepicted in FIG. 4B. The pseudo-noise sequence thus generated will berepeated in a cyclic fashion after a given number of values.Alternatively, the amplitude of the RF pulses can be modulated betweenOFF and ON as depicted in FIG. 4C. The acquisition of the response andphase cycling follow standard procedures. A Hadamard transform of theresponse produces the FID which, upon complex Fourier transform, yieldsa spectrum or a single projection when gradients are present.

FIG. 5 is a flow chart depicting the steps required to utilize thesystem of FIG. 1 to perform in vivo imaging of a sample.

The sample is placed in the resonator 50 and fields are set up 52, 54 tocause the molecules to be imaged to resonate at a selected low frequencyof about 300 MHz.

In many cases, a paramagnetic probe may be injected 56 into the sampleto improve imaging parameters. For example, if oxygen tension of thesample is to be measured the paramagnetic probe selected will interactwith oxygen to increase the relaxation rate. If short-lived freeradicals are to be imaged a spin trapping agent may be injected.

Subsequently, data acquisition will be started 58. A series of 10 to 60nanosecond RF pulses having a repetition rate of 4 to 5 microsecondswill be used to induce resonance in the sample. The receiver arm, gatedby pulses from the pulse generator 6, will detect, amplify, demodulate,sample, digitize, and sum, EPR parameters in the time periods between RFpulses.

Various projections will be imaged by changing the gradient field 60, 62and then image processing will be started 64 and the acquired image willbe displayed or printed 66.

Similar steps (excluding the injection of a probe), utilizing smallresonators and large gradients, can be used to perform FT EPRmicroscopy, especially in devices involving distribution of paramagneticcenters, such as semiconductor wafers, Lagmuir-Blodget films, qualitycontrol of conducting polymers and nondestructive determination ofstress or deterioration of polymeric substances in industry, commercial,and biomedical environment.

What is claimed is:
 1. A fast response pulsed radiofrequency (RF)electron paramagnetic resonance (EPR) system, with the system utilizinga system clock signal, comprising:a pulse generating sequential,non-overlapping transmit, diplexer, and receive gating pulses anultra-fast excitation pulse forming subsystem including:an RF signalgenerator for providing an RF signal having a frequency of between about200 MHz and about 400 MHz; a beam splitter, coupled to the output of theRF signal generator for splitting said RF signal into a reference RFsignal and an excitation signal RF signal; a phase shifter, coupled tosaid beam splitter to receive said transmitted RF signal, forcontrollably either passing or phase-shifting said RF excitation signalby 180°; a gating circuit, coupled to said phase shifter and including agate coupled to receive a transmit gating pulse from said pulsegenerator having a duration of about 10 to 90 nanoseconds, fortransmitting a received RF excitation signal when said transmit gatingpulse is asserted, to form an excitation pulse having a duration ofabout 10 to about 90 nanoseconds with rise times of less than about 2nanoseconds; an ultra-fast data acquisition system including:a gatedpreamplifier, having a signal input port and having a control inputcoupled to receive a receive gating pulse, said gated preamplifieramplifying RF radiation received at said signal input port only whensaid receive gating pulse is received and said gated preamplifier beingisolated from RF radiation received at said signal input port when saidreceive gating pulse is not received, with said gated preamplifier foramplifying EPR response RF radiation received at said signal input portto form an EPR response signal; demodulating means, coupled to receivesaid reference RF signal and said EPR response signal, for demodulatingsaid EPR response signal to form an EPR parameter signal; an ultra-fast,sampling and summing unit, coupled to said demodulating means, foraveraging a series of EPR parameter signals to increase signal to noiseratio, said sampling and summing unit including a high-speed sampler todigitize each received EPR parameter signal and a summing means, coupledto receive each digitized EPR parameter signal, for generating a runningsum of said digitized EPR parameter signals; a resonator for inducingparamagnetic resonance in a sample when an excitation pulse is received,for detecting EPR response RF radiation emitted from the sample due toparamagnetic resonance, and for outputting EPR response RF radiation; adiplexer, coupled to said pulse generator to receive said excitationpulse, coupled to said resonator to receive the EPR response RFradiation, coupled to the signal input port of said gated preamplifier,and having a control input for receiving a diplexer gating pulse of apreset duration, said diplexer for coupling said ultra-fast pulseforming subsystem to said resonator when said diplexer gating pulse isreceived, for isolating said pulse forming system from said ultra-fastdata acquisition system when said diplexer gating pulse is not received,and for providing said EPR response RF radiation from the resonator tothe input signal port of said gate preamplifier subsequent to receivingsaid diplexer gating pulse.
 2. The system of claim 1 wherein saidresonator is characterized by a Q parameter, where the bandwidth of theresonator response is inversely-proportional to the magnitude of Q andthe resonator ring-down time is proportional to Q, said system furthercomprising:Q-switching means, coupled to said resonator and said timingcontroller to receive a Q-switching pulse, for increasing resonator Qand decreasing ring-down time for said resonator when a Q-switchingpulse is asserted; and wherein said pulse generator generates aQ-switching pulse of about 20 nanoseconds immediately after saidtransmit pulse is received at said resonator.
 3. The system of claim 1further comprising:a DC magnet field for generating a constant magneticfield to induce magnetization in said sample; a gradient magnet forforming a gradient in said constant magnetic field.
 4. A method formeasuring EPR parameters utilized to perform in vivo measurement orimaging of oxygen tension in a living sample, with a gated RF amplifierfor amplifying response radiation generated by the sample, said methodcomprising the steps of:providing a paramagnetic contrast agent whichinteracts with in vivo oxygen in the living sample to increaserelaxation rate to improve imaging of oxygen; introducing saidparamagnetic contrast agent into a living sample to be imaged; providinga magnetic resonator; placing said living sample within the magneticresonator; generating a first series of RF excitation pulses, having anRF frequency between about 200 and 400 MHz separated by time intervalsgreater than about 4 microseconds; coupling each RF excitation pulse insaid first series to said resonator to induce EPR in said sample whileisolating the gated RF amplifier from said resonator; coupling saidgated RF amplifier to said resonator when said response radiation isgenerated in response to each excitation pulse in said first series togenerate a first series of corresponding EPR response signals based onthe interaction of in vivo oxygen with said paramagnetic contrast agentin time intervals between said first series of RF excitation pulses;digitizing and summing said first series of EPR response signals toobtain accurate values of EPR response signals; and processing saidaccurate value of said EPR response signals to generate a first seriesof EPR parameter signals.
 5. The method of claim 4 further comprisingthe steps of:generating a second series of RF excitation pulsesseparated by time intervals greater than about 4 microseconds;phase-shifting said second series of RF excitation pulses by 180° togenerate phase-shifted pulses; coupling each-phase shifted RF excitationpulse in said second series to said resonator to induce EPR in saidsample while isolating said gated RF amplifier from said resonator;coupling said gated RF amplifier to said resonator when said response RFradiation is generated in response to each phase-shifted pulse in saidsecond series to generate a second series of corresponding EPR responsesignals based on the interaction of in vivo oxygen with saidparamagnetic contrast agent in time intervals between said RF excitationpulses in said second series; digitizing and subtracting said secondseries of EPR response signals from said first series of EPR responsesignals to subtract systematic noise and DC bias to obtain accuratevalues of said EPR response signals; and processing said accurate valuesof said EPR response signals to generate a second series of EPRparameter signals.
 6. The method of claim 4 further comprising the stepsof:generating a first gradient magnetic field along a first axis priorto generating said first series of RF excitation pulses and maintainingsaid field until after said first series of EPR response signals havebeen generated to form a first projection of said sample; and generatinga second gradient magnetic field along a second axis; generating asecond series of RF excitation pulses, subsequent to generating thesecond gradient magnetic field, separated by time intervals greater thanabout 4 microseconds; coupling each RF excitation pulse in said secondseries to said resonator to induce EPR in said sample while isolatingsaid gated RF amplifier from said resonator; coupling said gated RFamplifier to said resonator when said response RF radiation is generatedto generate a corresponding second series of EPR response signals, basedon the interaction of in vivo oxygen with said paramagnetic contrastagent, in time intervals between said RF excitation pulses in saidsecond series; digitizing and subtracting said second series of EPRresponse signals from said first series of EPR response signals tosubtract systematic noise and DC bias to obtain accurate values of saidEPR response signals; and processing said accurate values of said EPRresponse signals to generate a second series of EPR parameter signalsand form a second projection of said sampler.
 7. A method for measuringEPR parameters utilized to perform pulsed EPR measurement or imaging ofa sample placed within a magnetic resonator which excites the samplewhen an RF radiation pulse is received to induce the sample to emitresponse RF radiation subsequent to excitation, with a gated RFamplifier for amplifying response RF radiation to form an EPR responsesignal, said method comprising the steps of:generating a correspondingfirst series of pseudo-random numbers having either a first or secondvalue; generating a first series of RF excitation pulses, having an RFfrequency between about 200 and 400 MHz and separated time intervalsgreater than about 4 microseconds; phase-shifting each pulse in saidfirst sequence by 180° if a corresponding number in said first serieshas said second value and transmitting each pulse without phase-shift ifa corresponding number in said first sequence is equal to said firstvalue to generate a first series of phase-processed excitation pulsespulses; coupling each phase-processed RF excitation pulse in said firstseries to said resonator to induce EPR in said sample while isolatingsaid gated RF amplifier from said resonator; coupling said gated RFamplifier to said resonator when said response RF radiation is generatedin response to each phase-processed pulse in said first series togenerate a corresponding first set of EPR response signals based onparamagnetic resonance in said sample in time intervals between saidfirst series of phase-processed RF excitation pulses; digitizing andsumming said first series of EPR response signals to obtain accuratevalues of said EPR response signals; and performing a Hadamardtransformation on obtained EPR response signals to obtain free inductiondecay parameters.
 8. A method for measuring EPR parameters utilized toperform in vivo measurement of free radicals in a sample comprising thesteps of:providing a living sample; providing a magnetic resonator;placing said living sample within the magnetic resonator; generating afirst series of RF excitation pulses, having an RF frequency betweenabout 200 and 400 MHz and separated time intervals greater than about 4microseconds; coupling each RF excitation pulse in said first series tosaid resonator to induce EPR in said sample while isolating the gated RFamplifier from said resonator; coupling said gated RF amplifier to saidresonator when said response radiation is generated in response to eachexcitation pulse in said first series to generate a first set ofcorresponding EPR response signals, based on in vivo paramagneticresonance of free radicals in said sample, in time intervals betweensaid first series of RF excitation pulses; digitizing and summing saidfirst series of EPR response signals to obtain accurate values of saidEPR response signals; and processing said accurate values of EPRresponse signals to generate a first series of EPR parameter signals. 9.The method of claim 8 further comprising the steps of:generating asecond series of RF excitation pulses separated intervals greater thanabout 4 microseconds; phase-shifting said second series of RF excitationpulses by 180° to generate phase-shifted RF excitation pulses; couplingeach-phase shifted RF excitation pulse in said second series to saidresonator to induce EPR in said sample while isolating said gated RFamplifier from said resonator; coupling said gated RF amplifier to saidresonator when said response RF radiation is generated in response toeach phase-shifted pulse in said second series to generate acorresponding second series of EPR response signals, based on in vivoparamagnetic resonance of free radicals in said sample, in timeintervals between said phase-shifted RF excitation pulses in said secondseries; digitizing and subtracting said second series of EPR responsesignals from said first series of EPR response signals to subtractsystematic noise and DC bias to obtain accurate values of said EPRparameters; and processing said accurate values of said EPR responsesignals to generate a second series of EPR parameter signals.
 10. Themethod of claim 8 further comprising the steps of:generating a firstgradient magnetic field along a first axis prior to generating saidfirst series of RF excitation pulses and maintaining said field untilafter said first series of EPR response signals have been generated toform a first projection of said sample; and generating a second gradientmagnetic field along a second axis; generating a second series of RFexcitation pulses, subsequent to generating the second gradient magneticfield, separated by time intervals greater than about 4 microseconds;coupling each phase shifted RF excitation pulse in said second series tosaid resonator to induce EPR in said sample while isolating said gatedRF amplifier from said resonator; coupling said gated RF amplifier tosaid resonator when said response RF radiation is generated to generatea corresponding second series of EPR response signals, based on in vivoparamagnetic resonance of free radicals in said sample, in timeintervals between said RF excitation pulses in said second series;digitizing and subtracting said second series of EPR response signalsfrom said first set of EPR response signals to subtract systematic noiseand DC bias to obtain accurate values of said EPR response signals; andprocessing said accurate values of said EPR response signals to generatea second series of EPR parameter signals and form a second projection ofsaid sample.
 11. The method of claim 8 further comprising the stepsof:providing a spin trapping agent; and introducing said spin trappingagent into said sample to stabilize said free radicals for imaging. 12.A method for measuring EPR parameters utilized to perform pulsed EPRmeasurement or imaging of a sample placed within a magnetic resonatorwhich excites the sample when an RF radiation pulse is received to emitresponse RF radiation subsequent to excitation, with a gated RFamplifier for amplifying response RF radiation to form an EPR responsesignal, said method comprising the steps of:generating a correspondingfirst series of pseudo-random numbers having either a first or secondvalue; generating a first series of RF excitation pulses, having an RFfrequency between about 200 and 400 MHz separated by time intervalsgreater than about 4 microseconds; modulating the amplitude each RFexcitation pulse in said first sequence to an OFF value if acorresponding number in said first series has said first value and to anON value if a corresponding number in said first sequence has saidsecond value to generate a first series of modulated RF excitationpulses; coupling each RF excitation pulse in said first series ofmodulated RF excitation pulses to said resonator to induce EPR in saidsample while isolating the gated RF amplifier from said resonator;coupling said gated RF amplifier to said resonator when said response RFradiation is generated in response to said first series of modulated RFexcitation pulses to generate a corresponding first set of EPR responsesignals based on paramagnetic resonance in said sample in time intervalsbetween said first series of modulated RF excitation pulses; digitizingand summing said first series of EPR response signals to obtain accuratevalues of said EPR response signals; and performing a Hadamardtransformation on obtained EPR response signals to obtain free inductiondecay parameters.
 13. A method for measuring EPR parameters utilized toperform RF FT EPR microscopy of free radicals or a paramagnetic contrastagent in a living sample placed within a magnetic resonator whichexcites the sample when an RF radiation pulse is received to emitresponse RF radiation subsequent to excitation, with a gated RFamplifier for amplifying response RF radiation to form an EPR responsesignal, said method comprising the steps of:generating a first series ofRF excitation pulses, having an RF frequency between about 200 and 400MHz separated by time intervals greater than about 4 microseconds;coupling each RF excitation pulse in said first series to said resonatorto induce EPR in said sample while isolating said gated RF amplifierfrom the resonator; coupling said gated RF amplifier to said resonatorwhen said response RF radiation is generated in response to eachexcitation pulse in said first series to generate generating a first setof EPR response signals based on in vivo paramagnetic resonance of freeradicals in said sample in time intervals between said first series ofRF excitation pulses; digitizing and summing said first series of EPRresponse signals to obtain accurate values of EPR response signals; andprocessing said first series of EPR response signals to generate a firstseries of EPR parameter signals.
 14. The method of claim 13 furthercomprising the steps of:generating a second series of RF excitationpulses separated by time intervals greater than about 4 microseconds;phase-shifting said second series of RF excitation pulses by 180° toform phase-shifted RF excitation pulses; coupling each phase-shifted RFexcitation pulse in said second series to said resonator to induce EPRin said sample while isolating the gated RF amplifier from saidresonator; coupling said gated RF amplifier to said resonator when saidresponse RF radiation is generated in response to each excitation pulsein said second series phase-shifted RF excitation pulses to generate asecond series of EPR response signals based on paramagnetic resonance ofin vivo free radicals in time intervals between said RF excitationpulses in said second series; digitizing and subtracting said secondseries of EPR response signals from said first series of EPR responsesignals to subtract noise and DC signals to obtain accurate values ofsaid EPR response signals processing said accurate values of said EPRresponse signals to generate a second series of EPR parameter signals.15. The method of claim 13 further comprising the steps of:generating afirst gradient magnetic field along a first axis prior to generatingsaid first series of RF excitation pulses and maintaining said fielduntil after said first series of EPR response signals have beengenerated to form a first projection of said sample; and generating asecond gradient magnetic field along a second axis; generating a secondseries of RF excitation pulses, subsequent to generating the secondgradient magnetic field, separated by time intervals greater than about4 microseconds; coupling each phase shifted RF excitation pulse in saidsecond series to said resonator to induce EPR in said sample whileisolating said gated RF amplifier from said resonator; coupling saidgated RF amplifier to said resonator when said response RF radiation isgenerated in response to each excitation pulse in said first second togenerate a corresponding second series of EPR response signals based onin vivo paramagnetic resonance of free radicals in said sample in timeintervals between said RF excitation pulses in said second series;digitizing and subtracting said second series of EPR response signalsfrom said first set of EPR response signals to subtract noise and DCsignals to obtain accurate values of said EPR parameters; and processingsaid accurate values of said EPR response signals to generate a secondseries of EPR parameter signals and form a second projection of saidsample.
 16. The method of claim 13 further comprising the stepsof:providing a spin trapping agent; introducing said spin trapping agentinto said sample to stabilize said free radicals for imaging.
 17. A fastresponse pulsed radiofrequency (RF) electron paramagnetic resonance(EPR) imaging system for forming an EPR image of a sample, said imagingsystem coupled to an RF signal generator that provides an RF excitationsignal and an RF reference signal, with the RF signal having a frequencyrange of about 200 to 400 Mhz and receiving a system clock signal, saidsystem comprising:a pulse generator, having an input coupled to receivethe system clock signal and said RF excitation signal, for generatingsequential, non-overlapping transmit, diplexer, receive, and Q-switchinggating pulses; a gating circuit, coupled to receive RF radiation andcoupled to receive a transmit gating pulse having a duration of about 10to 90 nanosecond, for transmitting said an RF signal when said transmitgating pulse is asserted, to form an RF excitation pulse having aduration of about 10 to about 90 nanoseconds with rise times of lessthan about 2 nanoseconds; an ultra-fast data acquisition systemincluding:a gated amplifier, having a signal input port and having acontrol input for receiving a receive gating pulse, said gated amplifierfor amplifying RF radiation received at said signal input port only whensaid receive gating pulse is received and said gated amplifier beingisolated from RF radiation received at said signal input port when saidreceive gating pulse is not received, with said gated amplifier foramplifying EPR response RF radiation received at said signal input portto form an EPR response signal; demodulating means, coupled to receivesaid EPR response signal and said RF reference signals, for demodulatingsaid EPR response signal to form an EPR parameter signal; an ultra-fast,sampling and summing unit, for averaging a series of EPR parametersignals to increase signal to noise ratio, said sampling and summingunit including a high-speed sampler to digitize each received EPRparameter signal and summing means, coupled to receive each digitizedEPR parameter signal, for generating a running sum of said digitized EPRparameter signals; and a resonator for inducing paramagnetic resonancein a sample when an excitation pulse is received, for detecting EPRresponse RF radiation emitted from the sample due to paramagneticresonance, and for outputting EPR response RF radiation; a diplexer,coupled to said to receive said RF excitation pulse, coupled to saidresonator to receive the EPR response RF radiation, coupled to thesignal input port of said gated amplifier, and having a control inputfor receiving the diplexer gating pulse of a preset duration, saiddiplexer for coupling said RF excitation pulse to said resonator whensaid diplexer gating pulse is received, for isolating said RF excitationpulse from said ultra-fast data acquisition system when said diplexergating pulse is not received, and for providing said EPR response RFradiation from said resonator to the input signal port of said gateamplifier subsequent to receiving said diplexer gating pulse; andQ-switching means, coupling said resonator to said diplexer and coupledto said pulse generating circuit to receive said Q-switching gatingpulse, for increasing resonator Q decreasing the ring-down time of theresonator.
 18. The system of claim 17 further comprising:a phaseshifter, coupled to receive said RF signal and having an output coupledto said gating circuit, for controllably either passing orphase-shifting said RF signal by 180°.